Abstract

Metastatic spread of cancer cells is the main cause of death of breast cancer patients,
and elucidation of the molecular mechanisms underlying this process is a major focus
in cancer research. The identification of appropriate therapeutic targets and proof-of-concept
experimentation involves an increasing number of experimental mouse models, including
spontaneous and chemically induced carcinogenesis, tumor transplantation, and transgenic
and/or knockout mice. Here we give a progress report on how mouse models have contributed
to our understanding of the molecular processes underlying breast cancer metastasis
and on how such experimentation can open new avenues to the development of innovative
cancer therapy.

Introduction

Breast cancer is the most frequently diagnosed form of cancer and the second leading
cause of death in Western women [1]. Death, and most of the complications associated with breast cancer, are due to metastasis
developing in regional lymph nodes and in distant organs, including bone, lung, liver,
and brain [1,2]. As in many other metastatic cancer types, specific molecular changes occurring within
both the tumor cells and the tumor microenvironment contribute to the detachment of
tumor cells from the primary tumor mass, invasion into the tumor stroma, intravasation
into nearby blood vessels or lymphatics, survival in the bloodstream, extravasation
into and colonization of the target organ and, finally, metastatic outgrowth [3,4].

In the recent past, our understanding of breast cancer progression and metastasis
has greatly profited from the use of genetically modified mouse models and advanced
transplantation techniques. Here we describe the currently employed mouse models of
breast cancer metastasis and how their use has contributed significantly to our understanding
of the molecular processes underlying breast cancer metastasis.

Mechanisms contributing to breast cancer metastasis

A critical step towards the generation of mouse models of breast cancer is the understanding
of the molecular pathways underlying mammary carcinogenesis. Our knowledge on how
breast tumor progression occurs has also been markedly improved by unraveling the
dynamics and the key factors of mammary gland development.

Mammary gland development

Mouse breast tissue undergoes continuous changes throughout the lifespan of reproductively
active females, mediated mainly by interactions between the mammary epithelium and
the surrounding mesenchyme (Figure 1). The mammary bud develops by forming a network of branched ducts invading into the
mammary fat pad [5]. With the release of ovarian hormones, terminal end buds are formed. They represent
the invading front of the ducts and they are able to proliferate, to extend into the
fat pad, and to form branches. During pregnancy and lactation, hormone-induced terminal
differentiation of the mammary epithelium into milk-secreting lobular alveoli takes
place. After weaning, the secretory epithelium of the mammary gland involutes into
an adult nulliparous-like state by apoptosis and redifferentiation. During these processes,
the developing mammary gland has the ability to induce angiogenesis to adjust for
blood supply and is protected against premature involution; it is therefore resistant
to apoptosis [6]. Interestingly, proliferation, invasion, angio-genesis, and resistance to apoptosis
are all features that are abused during the etiology of breast carcinogenesis.

Transformation and metastasis

Mammary gland morphogenesis and branching involve the regulatory function of several
signaling pathways, including signaling by Wnt family members [7], transforming growth factor-β (TGF-β) [8], insulin-like growth factor-I (IGF-I) [9], and epidermal growth factor (EGF) and others [10]. These pathways are frequently activated during the tumorigenic process by mutation
or gene amplification, thus allowing the mammary epithelium to expand, proliferate,
and invade neighboring tissue. The cross-talk and interactions between tumor cells
and the surrounding stroma, the extracellular matrix (ECM), and infiltrating cells
of the immune system are constantly modulating tumor development. The mammary stroma,
composed of pre-adipocytes, adipocytes, fibroblasts, endo-thelial cells, and inflammatory
cells, contributes functionally to mammary gland development [6]. In a similar manner, tumor–stroma interactions, occurring via soluble growth factors,
cytokines and chemokines, remodeling of the extra-cellular matrix, or direct cell–cell
adhesion, are critical for tumor growth, migration, and metastasis. Alteration of
the expression or function of adhesion molecules responsible for the adhesion of breast
cancer cells to themselves, to stromal cells, or to tumor matrix, including integrin
family members, immunoglobulin-domain cell adhesion molecules (such as L1 and NCAM),
cadherin family members, or other cell surface receptors (such as CD44), contributes
predominantly to late-stage tumor progression and metastatic dissemination of cancer
cells [11,12].

The formation of new blood vessels (angiogenesis) is crucial for the growth and persistence
of primary solid tumors and their metastases, and it has been assumed that angiogenesis
is also required for metastatic dissemination, because an increase in vascular density
will allow easier access of tumor cells to the circulation. Induction of angiogenesis
precedes the formation of malignant tumors, and increased vascularization seems to
correlate with the invasive properties of tumors and thus with the malignant tumor
phenotype [13]. In fact, angiogenesis indicates poor prognosis and increased risk of metastasis
in many cancer types, including breast cancer [14]. With the recent identification of lymphangiogenic factors and their receptors it
has also been possible to investigate the causal role of lymphangiogenesis in the
metastatic process (reviewed in [15]). It is therefore not surprising that molecules essential for mammary gland development,
many of them stromal factors, are also critical participants in breast carcinogenesis.

The knowledge gained on the several mechanisms contributing to tumor progression can
be used to design and generate better mouse models. At the same time, such models
allow a thorough investigation of all different aspects of multistage breast carcinogenesis,
including the genetic alterations leading to tumor onset, neovascularization, tumor
progression, and formation of metastasis in secondary organs.

Breast cancer metastasis models

Tumor transplantation

There are various ways to mimic breast cancer growth and metastasis in tumor transplantation
experiments. The site of injection, together with the specific tropism of the chosen
breast cancer cell line used, largely defines primary and secondary metastatic growth.
Orthotopic or ectotopic implantation of cancer cells in the skin or mammary fat pad,
with the formation of primary tumors and the subsequent formation of metastasis, in
part resembles the multiple stages involved in malignant breast cancer development
in patients [16]. In contrast, tail vein injection results mainly in lung metastasis, whereas portal
vein injection provokes colonization of the liver, and intracardiac infusion gives
rise to a broader target organ spectrum, including bone. Notably, the direct introduction
of cancer cells into the blood circulation should be considered an assay of organ
colonization and not a true metastatic process.

Depending on the species or genetic background of donor and host, syngeneic or xenograft
tumor transplantations need to be distinguished. Transplantation of cancer cells from
one mouse into another mouse with identical genetic backgrounds (syngeneic transplantation)
bypasses the immunologic host-versus-graft reaction and concomitantly allows the investigation
of the contribution of an intact immune system to malignant tumor progression [17,18]. Syngeneic mouse models have been employed to establish organ-specific metastasis
models by several rounds of transplantation/metastasis formation and the selection
of metastatic cell lines in vivo [19]. For example, 4T1 cells, which originally derive from a spontaneous mouse mammary
tumor of a BALB/C mouse, grow rapidly when injected into the fat pad of a syngeneic
animal and metastasize to lungs, liver, bone, and brain [19,20]. Sublines of 4T1 cells, which exhibit various degrees of metastatic dissemination,
have been employed recently to generate distinct gene expression signatures for each
stage of tumor progression, namely primary tumor formation, lymph node colonization,
metastatic outgrowth in the lymph node, and distant organ metastasis. These experiments
led to the identification of the transcriptional repressor Twist, some members of
the cadherin family of cell–cell adhesion molecules, and various chemokines as critical
factors in the distinct stages of metastatic tumor progression [20]. This and other syngeneic mouse models have also been successfully employed for the
testing of experimental drugs designed to interfere with tumor malignancy [18,21].

To investigate the growth and metastasis of human breast cancer cell lines in vivo, xenograft transplantation experiments are performed in immunocompromised mice [22]. Human breast cancer cells can be injected subcutaneously, intravenously, intracardially,
or orthotopically into the fat pad of the mouse [23]. For example, MDA-MB-231 cells, an estrogen-independent breast cancer cell line derived
from the pleural effusion of a cancer patient, is able to colonize bone, liver, lung,
adrenal glands, ovary, and brain after intravenous injection [24]. This cell line and organ-specific metastatic variants thereof have recently been
used to identify and functionally implicate a number of genes in organ-specific metastasis,
including IL-11, osteopontin and the connective tissue growth factor (CTGF) in osteolytic
metastasis [25,26], and epiregulin, CXCL1, matrix metalloproteinase-1 (MMP-1), cyclo-oxygenase-2 (COX-2),
inhibitor of differentiation-1 (Id1) and others in lung metastasis [27] (see below).

The implantation of established cell lines derived from human breast cancer is relatively
simple and allows the genetic or pharmacological manipulation of the implanted cells.
However, there are clear limitations to xenograft models. First, immune responses,
which have a key role during tumor development, are impaired in immunocompromised
mice. Second, stromal components are not of tumor origin. For example, carcinoma-associated
fibroblasts derived from a breast cancer patient support the growth of a breast carcinoma
cell line better than the normal tissue in a xenograft mouse co-implantation model.
Carcinoma-associated fibroblasts seem to activate and sustain CXCR4/stromal cell-derived
factor (SDF-1)-mediated chemokine signaling and to recruit endothelial progenitors
to the growing tumor, thereby promoting angiogenesis [28,29]. Last, human cells are apparently not fully adapted to grow in a murine environment.
For example, breast cancer metastasis to bone has recently been investigated in an
experimental mouse system in which both the breast cancer cells and the metastatic
target organ, the bone, are of human origin [30]. After orthotopic injection, cancer cells predominantly colonize the bone of human
origin, thus exhibiting a species-specific osteotropism.

Genetically modified mice

Several promoters can be used to drive the expression of transgenes in the mammary
epithelium (Table 1), and many known oncogenes have been expressed under their control to initiate or
modulate breast carcinogenesis in mice, including ErbB2/Neu, polyoma middle T antigen
(PyMT), simian virus 40 (SV40) T antigen, Ha-Ras, Wnt-1, TGF-α, and c-Myc. MMTV-Neu
and MMTV-PyMT transgenic mice (in which the expression of the oncogene is driven by
the Mouse Mammary Tumor Virus promoter) develop metastasis in lung and lymph nodes,
mainly after their first pregnancy, while other transgenic mice have to be combined
to generate double-transgenic mice that efficiently develop malignant cancers [31-35]. C3(1)-SV40 T-antigen transgenic mice develop invasive mammary carcinomas independently
of hormone supplementation or pregnancy, with a 15% incidence of lung metastasis.
This model recapitulates the loss of estrogen receptor-α expression that is frequently
observed in human breast cancer [36]. The most commonly used transgenic mouse models that develop metastatic mammary cancer
are summarized in Table 2.

Investigating the functional role of distinct genes during the multiple stages of
breast carcinogenesis requires the ability to modulate their function in time and
space [37]. Inducible transgene expression can be obtained by the use of the bacteria-derived
tetracycline-inducible system permitting the switching on or off (Tet-On/Tet-Off system)
of a gene of interest in a tissue- and time-specific manner [38]. In contrast, mice are modified by the genetic ablation of a gene of interest in
an inducible manner to generate conditional knockouts with the use of the Cre/loxP
phage recombinase system, for example [39]. To ablate a gene at a certain time point in mammary epithelial cells, recombinase
activity can be controlled by the expression of a tamoxifen-inducible version of Cre
(MMTV-ER™-Cre) or by using the tetracycline-inducible system to drive Cre expression
[40].

First comparisons of gene expression profiles obtained from mammary gland tumor models
initiated by different oncogenes have revealed several common and oncogene-specific
targets and similarities with human molecular breast cancer pathology [41]. The challenge now is to test whether genes identified in gene expression profiling
experiments with patient samples are able to modulate breast carcinogenesis in transgenic
mouse models, for example in the well-characterized MMTV-Neu and MMTV-PyMT mouse models
of breast carcinogenesis or in improved versions of these.

MMTV-Neu

Amplification of the gene encoding ErbB2, a member of the EGF receptor gene family,
is associated with 15 to 20% of human breast cancers, and in about 30% of cases the
increased expression of an activated form of ErbB2 is detected. Consistent with this
notion is the observation that transgenic expression of an activated form of the rat
homolog of ErbB2 (Neu) in MMTV-Neu transgenic mice results in the development of multifocal
adenocarcinomas with lung metastases at about 15 weeks after pregnancy [42]. Transgenic expression of wild-type ErbB2 in mammary gland also provokes tumor formation
and metastatic dissemination, yet with longer latency.

Doxycycline-inducible expression of ErbB2 in mammary epithelial cells of transgenic
mice also results in invasive mammary carcinoma and extensive metastasis, yet the
tumors regress with the loss of ErbB2 expression upon the withdrawal of doxycycline.
However, most mice exhibit recurrences of the tumors [43]. These recurrent tumors exhibit epithelial-mesenchymal transition (EMT), which seems
to be mediated by the upregulated expression of the transcriptional repressor Snail,
a molecular process that seems to have a high prognostic value in predicting human
breast cancer recurrence. Expression of oncogenic versions of ErbB2 that bind either
Grb-2 or Shc demonstrate that focal mammary tumors with a high rate of lung metastasis
require Grb-2-mediated signaling, whereas low metastatic multifocal mammary tumors
rely on Shc function [44].

MMTV-PyMT

Mammary gland-specific expression of PyMT under the control of the MMTV promoter/enhancer
in transgenic mice (MMTV-PyMT) results in widespread transformation of the mammary
epithelium and in the development of multifocal mammary adenocarcinomas and metastatic
lesions in the lymph nodes and in the lungs [45]. Tumor formation and progression in these mice is characterized by four stages: hyperplasia,
adenoma/mammary intra-epithelial neoplasia, and early and late carcinoma [46]. The close similarity of this model to human breast cancer is also exemplified by
the fact that in these mice a gradual loss of steroid hormone receptors (estrogen
and progesterone) and β1-integrin is associated with overexpression of ErbB2 and cyclin
D1 in late-stage metastatic cancer [47]. The MMTV-PyMT mouse model of breast cancer is furthermore characterized by short
latency, high penetrance, and a high incidence of lung metastasis occurring independently
of pregnancy and with a reproducible kinetics of progression.

In MMTV-PyMT transgenic mice, increased metastatic potential has been shown to depend
on the presence of macrophages in primary tumors and on the establishment of a chemoattractant
paracrine loop of colony-stimulating factor-1 (CSF-1) and EGF ligands between macrophages
and tumor cells [48,49]. In MMTV-PyMT/CSF-1-/- mice, tumor progression and metastasis are significantly delayed but restored on the
overexpression of CSF-1 in the mammary gland [48,50]. The crucial role of macrophages in sustaining tumor progression was further shown
by depletion of plasminogen, a downstream effector of CSF-1, either by its genetic
ablation or by affecting the expression of its activator uPA, resulting in significantly
reduced metastasis in the MMTV-PyMT mouse model without affecting primary tumor growth
[51,52]. The uPA/plasminogen system may contribute to metastasis mainly by ECM degradation.
The relevance of this mechanism is further supported by experiments with MEKK1-deficient
MMTV-PyMT mice, which show a significant delay in lung metastasis, whereas no differences
are observed in the primary tumor growth. MEKK1 signaling is involved in cell adhesion
and controls uPA induction. Accordingly, MEKK1-deficient mice display decreased levels
of uPA, which result in reduced levels of activated plasminogen and impaired tumor
cell migration and invasiveness [53].

The role of adhesion molecules during mammary gland tumor progression has also been
addressed with the use of MMTV-PyMT mice. Specifically, loss of CD44 promotes lung
metastasis in these mice, highlighting the role of tumor–stroma interaction for adhesion
and invasion [12]. CD44 expression on tumor cells mediates their interaction with hyaluronan-expressing
stromal cells and results in increased cancer progression. Loss of another adhesion
molecule, Muc-1, in the MMTV-Wnt1 tumor model results in a delayed onset of tumorigenesis
as well as delayed metastasis to lungs. Muc-1 seems to form complexes with β-catenin
at the cell membrane and in the cytoplasm of cells at the tumor's invading front [54].

Recent results indicate that changes in cell adhesion have a critical function in
tumor progression [11]. For example, the epithelial adherens junction molecule E-cadherin is considered
a tumor and invasion suppressor. Forced expression of E-cadherin prevents tumor cell
migration and invasion, whereas inhibition of E-cadherin function enhances tumor cell
invasion and metastatic dissemination. E-cadherin is irreversibly lost in more than
85% of invasive lobular breast cancer associated with an invasive phenotype, and in
the remaining 15% the retention of E-cadherin is associated with dysfunctional adhesion.
Interestingly, a transgenic mouse model of epithelial loss of both E-cadherin and
p53 develops metastatic mammary carcinoma resembling human invasive lobular breast
cancer (J. Jonkers, personal communication). Taken together, these examples indicate
that transgenic mouse models of breast cancer metastasis are essential to understanding
the role of several molecules in modulating key steps during malignant progression.

In vivo imaging

Non-invasive in vivo imaging techniques have been developed to reveal metastatic mammary tumors in experimental
systems. Cell lines and transgenic mice can be engineered to express luminescent or
fluorescent markers, permitting the visualization of primary tumor growth and the
formation of metastatic nodes in live animals over time. MMTV-enhanced green fluorescent
protein (eGFP) mice or mice in which expression of eGFP or luciferase marker genes
is 'switched on' in the mammary gland in a Cre-dependent way upon crossing with either
WAP-Cre or MMTV-Cre mice have been generated [55-57]. Tumor growth and metastasis formation can be easily monitored in composite transgenic
animals after crossing of these mice with breast cancer mouse models [58]. Moreover, tumor progression and the actual metastatic mobility of tumor cells can
be detected in live animals by multiphoton microscopy, positron-enhanced tomography
scans, and magnetic resonance analysis [59-61]. Furthermore, the newest technologies, including intravital microscopy [62,63], in vivo flow cytometry [64], and multicolor fluorescent-based approaches, provide the possibility of quantitatively
detecting individual tumor cells in living animals and documenting their clearance,
motility, and migration to or retention in target organs.

Molecular pathways dissected using breast cancer mouse models

Transforming growth factor-β

TGF-β exerts a dual role during tumor progression: by inducing the expression of cell
cycle inhibitors, it acts as a tumor suppressor during the initial phases of tumor
progression. Yet it promotes metastasis and invasion in the later stages by inducing
EMT [8]. The role of TGF-β in breast cancer metastasis is still under investigation. One
of its major functions, beside the induction of EMT, is inducing the migration and
intravasation of breast cancer cells into the circulation, thereby promoting osteolytic
metastasis [65]. Expression of TGF-β1 in double-transgenic MMTV-Neu/MMTV-TGF-β1 mice increased the
number of cancer cells circulating in the blood as well as the lung metastases, whereas
primary tumors developed at unchanged frequency [66,67]. Inducible expression of TGF-β1 in mammary glands of MMTV-PyMT transgenic mice also
demonstrated the pro-metastatic function of TGF-β1 [68]. Transgenic mice expressing TGF-βRI or a dominant-negative version of TGF-βRII under
the control of the MMTV promoter crossed with MMTV-Neu mice promoted and repressed,
respectively, tumor metastasis [44]. Surprisingly, conditional knockout of TGF-βRII in the mammary epithelium of the
MMTV-PyMT mouse resulted in increased metastasis formation [69]. Together, these experiments in mouse models demonstrate the pivotal role of TGF-β
signaling in breast carcinogenesis. These observations have implications for the development
of anti-metastatic therapies. For example, long-term treatment of MMTV-Neu mice with
a soluble version of TGF-βRII protects MMTV-Neu mice from metastasis without increasing
primary tumor growth, hence selectively blocking the metastatic effects of TGF-β while
not affecting its functions in early tumor stages [70]. Chronic exposure to the soluble TGF-βRII in these mice did not cause any unwanted
side effects, suggesting a potential avenue for the development of therapy. Small
inhibitors of the TGF-β receptor kinase activity and agents specifically blocking
TGF-β-mediated signaling pathways are currently in clinical trials [71].

EGF family members

The importance of TGF-α, an EGF family member, in mammary tumor onset has been demonstrated
by the transgenic expression of TGF-α under the control of several mammary epithelium-specific
promoters. Such tissue-specific expression has led to distorted mammary gland development.
However, primary tumors and pulmonary metastasis formed only after the combination
of several additional tumor-promoting factors, such as crossing TGF-α transgenic mice
with MMTV-Myc transgenic mice or treating MMTV-TGF-α mice with chemical carcinogens.
In double-transgenic MMTV-TGF-α; MMTV-TGF-β mice, tumor development is, however, suppressed
[72].

We have already introduced the importance of ErbB2 in breast carcinogenesis. In addition,
amplification of the gene encoding EGFR correlates with increased metastasis and is
a bad prognosis factor in breast cancer [73]. MMTV-Neu mice have also been extensively employed to investigate the functional
contribution of EGFR to mammary carcinogenesis. EGFR-mediated signaling contributes
to invasion, intra-vasation and metastasis, along with the mitogenic signaling in
this model [49,74,75]. Moreover, EGFR contribution to metastasis was shown by using MTLn3 rat mammary adeno-carcinoma
cells injected into the fat pad of mice. By quantifying the number of tumor cells
in the blood as a direct measure of cell intravasation it was possible to show that
EGFR acts via increased cell motility and intravasation rather then by affecting cell
proliferation [76]. A neutralizing antibody against ErbB2 (Herceptin) has been developed to repress
the tumorigenic stimuli of ErB2 and has been approved for clinical use (reviewed in
[10]). Together with newly developed inhibitors of EGFR signaling, combinatorial repression
of EGFR and ErbB2 activity may therefore be an efficient way to combat breast cancer.

Wnt signaling

Wnt family members were the first proto-oncogenes to be discovered by an MMTV-mediated
insertion–activation mechanism. Transgenic expression of Wnt-1 in the mammary gland
of transgenic mice results in mammary adeno-carcinomas with metastasis to lymph nodes
and lungs [7]. Moreover, Wnt-1 collaborates with fibroblast growth factor-3, another MMTV-insertion-activated
gene, in tumor onset. Surprisingly, in double-transgenic MMTV-Wnt-1;MMTV-TGF-β animals,
tumor cell proliferation is not repressed by TGF-β expression, showing an opposite
effect to that observed for MMTV-TGF-α; MMTV-TGF-β mice (see above) [77].

Genes involved in organ-specific metastasis

Cancers developing in a certain organ usually exhibit particular patterns of organ-specific
metastasis. Breast cancer predominantly colonizes bone, followed by axillary and other
lymph nodes, lung, liver, brain, and (rarely) adrenal glands. A combination of physical
factors, such as lymphatic and blood vessel capillary networks encountered by disseminating
tumor cells, and environmental factors, such as chemo-attractive cytokines or chemokines
and the presence of 'vasculature addresses', contribute to the specific dissemination
of metastastic cancer cells [78,79]. One possible underlying mechanism is that breast cancer cells follow a cytokine
gradient by co-opting immune cells' strategies to arrive at target organs [80].

Xenograft transplantation experiments using the MDA-MB-231 cell line have been instrumental
in demonstrating the functional role of certain genes in organ-specific breast cancer
metastasis. For instance, prevention of CXCR4 expression by using short interfering
RNA technology or blocking its function with specific antibodies or synthetic peptides
repressed the formation of lung metastasis, indicating that the CXCR4 ligand, SDF-1,
expressed by metastatic target organs, is recruiting tumor cells via CXCR4, which
is expressed on breast cancer cells [80-82]. Orthotopic, intracardiac, and tail vein injections of MDA-MB-231 cells have also
been performed to identify genes modulating organ-specific metastasis, for example
to bone or lung [25,27]. Gene expression profiling experiments with sublines of MDA-MB-231 selected for organ-specific
metastasis have identified specific gene expression signatures for different organ-specific
metastases. The functional involvement of these genes and factors in directing organ-specific
metastasis was demonstrated subsequently. Genes involved in lung metastasis include
those encoding the EGF-like factor epiregulin, CXCL1, MMP-1 and MMP-2, SPARC, vascular
cell adhesion molecule-1 (VCAM-1), Id1, and COX-2, and genes promoting bone metastasis
include those encoding IL-11, osteopontin, CTGF, CXCR4, and MMP-1 [25,27].

Overexpression of osteopontin induces metastasis of poorly metastatic MDA-MB-231 cells,
whereas its downregulation is correlated with reduced osteolytic metastasis [26]. Osteo-pontin upregulates uPA plasminogen activator, which, upon binding to integrins
and surface receptors, provokes the activation of both the hepatocyte growth factor
(HGF) and EGF pathways [83]. Xenograft transplantation of MT2994 primary breast cancer cells has shown that the
expression of osteopontin was associated with a constitutive activation of the phosphoinositide
3-kinase pathway and a metastatic phenotype of tumor cells [74]. Moreover, osteopontin can induce the expression of alternatively spliced isoforms
v6 and v9 of CD44 in breast cancer cells, leading to an increase in cell migration
[84].

In a similar approach, sublines of the breast cancer cell line MDA-MB-435 have been
selected for specific colonization of lung, lymph node, and thorax. Several adhesion
and matrix molecules are correlated with lymph node metastasis, including CD73, a
cell surface protein previously implicated in lymphocyte homing to lymph nodes [85]. Moreover, MDA-MB-468 variant metastatic cells with tropism to lymph nodes may use
differential expression of adhesion molecules and may mimic angiogenesis pathways
to reach lymph nodes [86]. Notably, these cells express α9β1 integrin, an integrin that is specifically expressed
on lymphatic endothelial cells and can bind many ligands previously implicated in
metastasis, including osteopontin, tenascin C, VCAM-1 and the lymphangiogenic factors
vascular endothelial growth factor (VEGF)-C and VEGF-D.

Recent work has documented a role for RANK/RANK ligand (RANKL) signaling together
with parathyroid hormone-related protein (PTHrP) and osteoprotegerin in bone metastasis.
Treatment with a humanized antibody against PTHrP significantly suppressed osteolytic
metastasis in mice injected with a subline of MDA-MB-231 that showed high metastatic
ability to bone and expressed high levels of PTHrP, IL-8, IL-6, and MMP-1 [87]. The importance of the role of RANK/RANKL signaling in the regulation of tumor cell
migration has also been reported for melanoma cells in vivo [88], whereas experiments performed with MDA-MB-231 breast cancer cells have shown that
the RANK soluble receptor, osteoprotegerin, is effective in specifically decreasing
bone metastasis by preventing the signaling that mediates the differentiation and
activation of osteoclasts [89]. However, in an intra-tibial ectotopic injection model of osteoprotegerin and PTHrP
overexpression by MCF-7 breast cancer cells it was revealed that overexpression of
osteoprotegerin by tumor cells actually supports tumor growth [90].

Upregulated expression of VEGF-C, and to a smaller extent that of VEGF-D, is highly
correlated with lymphangiogenesis and lymph node metastasis in cancer patients. Moreover,
forced expression of VEGF-C or VEGF-D in tumor cell lines or in transgenic mouse models
of tumorigenesis results in upregulated lymphangiogenesis and in the formation of
lymph node metastasis [15]. The role of lymphangiogenesis and angiogenesis in breast cancer metastasis is a
major focus of current research. Mammary overexpression of the blood vessel angiogenic
factor VEGF-A markedly accelerates the formation of lung metastasis in MMTV-PyMT mice,
not only by promoting tumor angiogenesis but also by sustaining tumor proliferation
and survival [91]. In a xenograft tumor transplantation model using MDA-MB-231 breast cancer cell line
variants with brain tropism, the formation of brain metastases seems highly dependent
on the presence of VEGF-A [92]. Moreover, in orthotopic xenograft transplantation of human breast cancer cells with
high or low metastatic ability (MDA-MB-435 and MCF-7, respectively), overexpression
of VEGF-C induces intra-tumoral lymphoangiogenesis and the subsequent formation of
lymph node and lung metastasis [93,94]. Blockade of VEGF receptor-3 signaling by specific antibodies inhibits regional and
distant lymph node metastasis in these models, whereas VEGF receptor-2 inhibition
results in a suppression of angiogenesis and tumor growth. Notably, a combination
of the two treatments suppresses the formation of metastases better than single treatments
[95]. These results indicate that angiogenic and lymphangiogenic factors may have central
roles in defining organ-specific breast cancer metastasis.

Conclusion

Elucidation of the molecular mechanisms underlying breast cancer progression and metastasis
has gained tremendously from mouse models in which the multiple stages of tumor progression
are recapitulated. However, despite their obvious convenience in basic cancer research
and in the testing of experimental therapies, the use of mouse models carries several
limitations. There are obvious differences between human and mouse tumorigenesis,
among which are the kinetics of carcinogenesis and the final size of tumors, differences
in cell intrinsic features such as the requirements to transform cells, and differences
in organ-specific gene expression, in physiology, metabolism, pathology, and in the
immune system. Moreover, metastatic dissemination occurs mainly via hematogenous spreading
to lungs and lymph nodes in MMTV-PyMT and MMTV-Neu mice, as opposed to the initial
spreading of cancer cells to local lymph nodes via the lymphatics in human breast
cancer.

Another important aspect to the understanding of breast cancer metastasis is the role
of different subpopulations of breast cells, including cancer stem cells. A great
effort is put into their isolation by means of molecular markers or functional assays.
The use of transplanted breast cancer stem cells isolated from mice harboring different
genetic modifications thereby offers a valuable tool not only in the unraveling of
breast cancer development but also in designing effective therapeutic strategies.

Recent technological advances have greatly improved the use of animal models in breast
cancer research, such as the use of bioluminescence and fluorescence systems, magnetic
resonance, positron-enhanced tomography scans or in vivo confocal analysis to image tumor development in live animals, also allowing observation
for long periods. Moreover, extended time-lapse observation of labeled tumor cells
in vivo provides new insights into the actual dynamics of tumor growth, extravasation, cell
migration, and organ colonization, as well as the contribution of the tumor stroma
and subsets of immune cells. Finally, gene expression analysis of tumor samples matched
with normal tissue from patients will provide gene signatures that will have to be
tested in vivo by proof-of-concept experiments in reliable mouse models of breast cancer metastasis.

In the future it will be necessary to generate mouse models that more accurately recapitulate
human breast carcino-genesis, while offering the advantages of model systems, such
as easy genetic or pharmacological manipulation and imaging. The quest for such improved
models has just begun.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

We are grateful to Dr Miguel Cabrita and Dr François Lehembre for critical comments
on the manuscript, and to Dr Jos Jonkers for sharing unpublished results. Research
in the laboratory of the authors is supported by the Krebsliga Beider Basel, Novartis
Pharma Inc., NCCR Molecular Oncology, the Swiss National Science Foundation and the
EU-FP6 framework programs LYMPHANGIOGENOMICS LSHG-CT-2004-503573 and BRECOSM LSHC-CT-2004-503224.